“Crotonate cultures were prepared in a similar manner substituting equimolar
amounts of crotonate for butyrate. Culture purity was checked by regular microscopic examination and inoculation of thioglycolate broth (Difco Laboratories, Inc., Detroit, MI, USA), which does not support the growth of either species.” “Generation times were calculated from the linear portion of the growth curve by measuring the time the culture took to double its optical density. Direct microscopic counts were done using a Petroff-Hausser counting chamber”. Growth was monitored by spectrophotometry at 600 nm. S. wolfei was grown in co-culture with M. hungatei and the “best” ratio was when grown with butyrate (1:3) compared with grown with only crotonate (1:47). After grown in co-culture, S. wolfei was then grown pure in agar plates, where it appeared as tiny opaque colonies of 1 mm od diameter (0.2% crotonate). After agar, pure cultres were grown with 0.2% crotonate and 0.3 Na2SO4 and grown was achieved within 360 h. Growth was observed as well without Na2SO4. Growth was inhibited by the presence of 20% of H2 in the headspace. When incubated with butyrate, growth wasn’t achieved unless co-cultured with M. hungatei or D. vulgaris (in the presence of sulfate*). Max absorbance was 0.47 when co-cultured. Max growth rate of pure S. wolfei was 0.029 1/h with 20-70 mM crotonate was used. Max PHB percentage obtained when grown pure was 20%. Gram-negative, slightly helical rods, 0.5 to 1.0 by 2.0 to 7.0 µm with slightly tapered rounded ends. Most cells occur singly or in pairs with helical chains of three or more often observed. Multiplication by binary fission. Cells possess two to eight flagella with a diameter of about 20 nm that are laterally inserted in a linear fashion on the concave side of the cell about 130 nm or more apart. Under most conditions, cells usually exhibit only a sluggish twitching motility.(McInerney, Bryant, Hespell, & Costerton, 1981)(Beaty & McInerney, 1987) Grows only with fatty acids of 4 to 8 carbons and produces propionate and acetate. Medium had originally rumen fluid (difficult stuff to get) but S.wolfei was able to be grown without it in a “new medium”. Medium was boiled under an 80% N2- 20% CO2 gas phase. Growth of 0.039 1/h and absorbance of 0.5 with rumen fluid. With the “new medium” the growth rate was 0.023 and absorbance od 0.08; however, adding lipoic acid, B12, B7 and B1 resulted in a similar growth as with rumen fluid. Max absorbance was reached at 311 h of incubation. No growth was observed before 192 h. p-aminobenzoic acid stimulated growth as well. The deletion of trace minerals (except FeSO4 and CoCl2*6H2O) in the defined medium with vitamins didn’t affect growth. (Beaty & Mcinerney, 1990) S. wolfei has two subspecies: wolfei and saponavida. S.wolfei wolfei degrades fatty acids with 4 to 8 carbons and S. wolfei saponavida degrades fatty acids with 4 to 18 carbons. Best growth rates occur at near neutral pH and with temperatures between 35-37 °C. The use of crotonate as substrate bypasses an unfavorable step of the oxidation of butyril CoA to crotonyl CoA. For each crotonate oxidized to acetate, another crotonate is reduced to butyrate. (Dworkin, Falkow, Rosenberg, Schleifer, & Stackebrandt, 2006) The substrate used to grow S. wolfei had 20 mM sodium crotonate, 0.4 mg/L of resarzurine, 200 µg/L of lipoic acid and 400 µg/L of thiamine. “Growth was monitored by determining the optical density (OD) against sterile medium. Prior to measurement, a few grains of sodium dithionite were added to the cuvettes to keep resazurine in its reduced state.” The end of exponential growth was achieved at 10 days when co-cultured. (Müller, Schleheck, & Schink, 2009)
Two thirds of the ATP made go
to the reverse electron transport and the other third goes to growth. The reverse electron transport produces H2 and formate. Electron transport by its pili wasn’t observed since S. wolfei lacks the genes for that. It lacks the genes for aerobic and anaerobic respiration. Stains for gram negative. Comes from the philum Firmicutes. Has motility (*) and probably has the capability of sporulating. Produces PHA while it is in exponential growth without being stressed by nutrient limitation, it does it by converting two molecules of acetyl-CoA to acetoacetyl- CoA and then to D(-)-3- hydroxybutyryl-CoA, or by using a b-oxidation intermediate to D(-)-3- hydroxybutyryl-CoA. The substrate used to grow S. wolfei had 20 mM sodium crotonate. (Sieber et al., 2010) References
Beaty, P. S., & Mcinerney, M. J. (1990). Nutritional Features of Syntrophomonas-Wolfei.
Applied and Environmental Microbiology, 56(10), 3223–3224. Beaty, P. S., & McInerney, M. J. (1987). Growth of Syntrophomonas wolfei in pure culture on crotonate. Archives of Microbiology, 147(4), 389–393. https://doi.org/10.1007/BF00406138 Dworkin, M., Falkow, S., Rosenberg, E., Schleifer, K.-H., & Stackebrandt, E. (Eds.). (2006). The Prokaryotes. New York, NY: Springer US. https://doi.org/10.1007/0-387-30744-3 McInerney, M. J., Bryant, M. P., Hespell, R. B., & Costerton, J. W. (1981). Syntrophomonas wolfei gen. nov. sp. nov., an Anaerobic, Syntrophic, Fatty Acid-Oxidizing Bacterium. Applied and Environmental Microbiology. Müller, N., Schleheck, D., & Schink, B. (2009). Involvement of NADH:acceptor oxidoreductase and butyryl coenzyme A dehydrogenase in reversed electron transport during syntrophic butyrate oxidation by Syntrophomonas wolfei. Journal of Bacteriology, 191(19), 6167–6177. https://doi.org/10.1128/JB.01605-08 Sieber, J. R., Sims, D. R., Han, C., Kim, E., Lykidis, A., Lapidus, A. L., … McInerney, M. J. (2010). The genome of Syntrophomonas wolfei: New insights into syntrophic metabolism and biohydrogen production. Environmental Microbiology, 12(8), 2289– 2301. https://doi.org/10.1111/j.1462-2920.2010.02237.x ……
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